Project Summary Ischemic stroke (IS), loss of cerebral blood flow due to occlusion of cerebral blood vessels, remains one of the most severe health problems in the world. Current treatments for stroke, such as thrombolytic agents, PTAS, and neuroprotective drugs have shown to be helpful; however, they are also limited by a narrow therapeutic time window, risk of re-stroke and low effectives. Looking for an ideal therapeutic strategy that protect brain from acute injury and promote angiogenesis and neurogenesis for neurological functional recovery, is thus imperative needed. Endothelial progenitor cells (EPCs) with the ability to differentiate into mature endothelial cells (ECs), are known to participate in angiogenesis and neurogenesis. EPC transplantation has shown promising results for IS therapy. Emerging evidence suggests that stem cell released EXs could be used for treating IS. EXs are endosomal origin small-membrane vesicles mediating cell-cell communication by transferring miRNAs (miRs). Stem cell EXs hold advantages over stem cells because of lacking self-proliferation and are easier to store and deliver than cells. In this project, we aim to use EPC-released EXs (EPC-EXs) as a novel avenue for IS therapy as they selectively carry enriched miR-126. We have found that EPC-EXs protect ECs from hypoxia/reoxygenation (H/R)-induced injury and dysfunction mainly through their carried miR-126; transfusion of EPC-EXs could preserve cerebral blood flow (CBF) and decrease infarct volume after IS. Of interest, the anti- apoptosis/anti-oxidative stress miR-210 has been found to be decreased in the stroke patients. We discovered that miR-210 primed EPC-EXs (EPC-EXsmiR210) are more effective than EPC-EXs in protecting ECs from H/R- induced injury. Taken together, by combining the beneficial effects of EPCs (stem cells), EXs (novel player of brain microenvironment), and miRs (multiple signaling pathway targets), we believe that EPC-EXsmiR210 could serve as a novel therapeutic avenue for IS. Three aims are designed to test our hypothesis that EPC-EXsmiR210 have enhanced therapeutic effects on IS by protecting brain cells from acute ischemic injury and promoting angiogenesis/neurogenesis for neurological functional recovery through EPC-EXs, miR-126 and miR-210 downstream pathways, VEGF/VEGFR2/PI3K/Akt, PIK3R2/PI3K/Caspase and BDNF/TrkB/PI3K/Akt. Aim 1: Determine whether EPC-EXsmiR210 have enhanced effects on protecting ECs and neurons from H/R-induced injury/dysfunction by targeting multiple pathways. Aim 2: Determine whether EPC-EXsmiR210 exert enhanced effects on protecting brain from IS-induced acute damage by targeting multiple pathways. Aim 3: Determine whether EPC-EXsmiR210 exert enhanced effects on promoting neurovascular genesis and functional recovery after IS by targeting multiple pathways. .